HIV/AIDS is a global pandemic with 35 million individuals living with HIV infection worldwide. The objectives of this project are to define the unique epidemiological, clinical, virologic, and immunologic features of HIV infection in developing countries, to determine the viral kinetics associated with sexual transmission, and to characterize the molecular strains of HIV internationally for infectiousness and progression of disease. HIV/AIDS is a global pandemic with 34 million individuals living with HIV infection worldwide. Accurate methods to estimate HIV incidence are needed to monitor the leading edge of the epidemic, identify groups at high risk of infection, and evaluate the effectiveness of prevention interventions. We have developed accurate multi-assay algorithm to estimate HIV incidence from cross-sectional surveys both in a US HIV-1 clade B epidemic and in a southern African HIV-1 clade A and C endemic area. In a US setting we demonstrated that these could accurately estimate incidence by comparing the incidence based on cross-sectional incidence testing at the end of in three observational cohorts to the incidence observed in those cohorts. We analyzed the performance of a new cross-sectional incidence assay, the Limiting Antigen avidity assay (LAg-avidity) and demonstrated that the assay could not be used as described by the CDC. After analyzing >500,000 potential algorithms using up to five different biomarkers, we identified optimal testing algorithm which used the LAg avidity. Using this algorithm we accurately estimated incidence compared to that observed in three longitudinal cohorts. In comparison with a vaccine preparedness cohort (HIVNET001) cohort study, annual incidence based on HIV seroconversion was 1.04% (95% CI: 0.70%-1.55%), while the incidence estimate obtained using the LAg-avidity based MAA was essentially identical: 1.09% (95% CI: 0.60%-1.84%). In a low incidence cohort of women in the United States (HPTN064) the observed incidence was 0.24% (95% CI: 0.07%-0.62%) while the MAA based incidence estimate was 0.26% (95% CI: 0.03%-0.95%). In a high incidence cohort of African American MSM, the observed incidence was 3.0% (95%CI 2.0 4.4%) while the MAA based estimate was 3.4% (95%CI 1.8% - 6.2%). In a southern African epidemic setting we analyzed the data from seven African cohorts (5,325 samples from 3,436 individuals with known duration of HIV infection 1 month to 10 years) and screened 403 different algorithms to identify the best algorithm to determine the primary outcome of Project Accept (HPTN 043). Project Accept is a cluster-randomized trial of 48 villages and > 50,000 individuals in four countries to determine if community-based voluntary counselling and testing reduced HIV incidence at a population level. This was the first large intervention trial that used cross-sectional incidence testing to measure its primary outcome and determined a 13.9% reduction in the intervention villages compared to the controls. We have also determined that individuals infected with different subtypes have different rates of antibody maturation, at least as measured by these cross-sectional incidence assays. Individuals infected with subtype D often fail to illicit highly avid antibody responses. We continued our studies of the molecular epidemiology of HIV focusing on Uganda and Malaysia. In Uganda we further investigated our previous findings that HIV-1 subtype D has much greater pathogenic effect than subtype A. By studying five different genomic regions among 496 seroconverters from the Rakai Health Sciences Program, we determined that having a subtype D polymerase had the greatest impact on disease progression. We also demonstrated that subtype D polymerases had much greater replication capacities than subtype A viruses and that those individuals infected with subtype D virus with high replication capacity had the greatest rate of disease progression. Furthermore we demonstrated that viral load and replication capacity were independent predictors of disease progression in this cohort. At a population level, using molecular, spatial and behavioral data, we demonstrated that the majority of incident infections acquired outside the household occur from outside the community, which suggest that HIV prevention efforts should be conducted at very broad geographic levels. We continued our research in Singapore where we had recently identified a new circulating recombinant form (CRF) of HIV, CRF51_01B. We determined that CXCR4 usage was common in CRF01_AE and CRF51_01B, which in part explains the increased rate of CD4 loss we see in individuals infected with these strains compared to subtype B in the US. We have expanded our use of ultra-deep pyrosequencing to identify HIV superinfection and describe its effects on the pandemic. As part of this research we established multiple collaborations to expand our work on HIV superinfection to explore its role on HIV transmission in the HPTN 052 and PEPI clinical trials, occurrence in seroconverters from the CAPRISA 004 clinical trial, and in a group of female sex workers in Kampala, Uganda. We have also identified the first case of superinfection with a known source partner from a group of virally discordant seroconcordant HIV+ couples from Masaka Uganda. In collaboration with Dr. John Mascola at the VRC we are planning on exploring the role of broadly neutralizing antibodies before and after HIV superinfection. We are also exploring this question in two other populations of MSM from Denmark and IV drug users from Baltimore MD. We have also begun collaborations in the US and in South Africa to examine the role of HIV superinfection and other HIV factors in HIV+ to HIV+ organ transplantation.